As the main component of cytoskeleton, microtubule has dynamic characteristics of polymerization and depolymerization, and plays an important role in maintaining the morphology of cells, division and proliferation of cells, composition and transport of organelles and conduction of semiochemicals. The antineoplastic drug targeting the microtubule is applied by making use of its dynamic characteristics, either by promoting or inhibiting its polymerization, so as to directly influence mitosis of cells and halt cell division in G2/M phase. Researches show that the microtubule has at least 3 different drug-binding sites: Taxol site, vincristin site and colchicine site. Taxol can inhibit depolymerization of tubulin and stabilize the structure of microtubule; and vinblastine and colchicine can inhibit polymerization of tubulin through respective sites of their actions.
Drugs used to inhibit depolymerization of microtubule, taking Taxol as a representative, are now widely applied for curing breast cancer, ovarian cancer, lung cancer, non-small cell lung cancer, etc. Drugs used to inhibit polymerization of microtubule have two different binding sites: colchicine site and vinblastine site. Drugs acting on the vinblastine site, taking vinblastine, vincristin, etc. as representatives, are now clinically applied to curing leukemia, lymphoma, non-small cell lung cancer, etc. Drugs acting on the colchicine site are represented by colchicine, Podophyllotoxin and Combretastatin (CA-4). Since the cavity volume of colchicine site is relatively small and its corresponding inhibitor structure is relatively simple, researches for its inhibitors have aroused much attention in recent years, and some derivatives for curing tumors have entered into clinical study and have demonstrated promising application prospects, such as E7010
ZD6126
etc. In the treatment of inflammatory diseases, colchicine has been clinically applied to curing acute gout and gouty arthritis. Due to the success achieved by applying taxol and vinblastine compounds for tumor treatment, scientists are rather confident in developing microtubule inhibitors targeting colchicine site.
Compared to the tumor drugs targeting taxol site and vinblastine site, the tumor drugs targeting colchicine site mainly have two important advantages. One advantage is that the chemotherapy drugs targeting colchicine site can not only inhibit polymerization of tubulin, but also generally inhibit generation of new vessels of tumors. Therefore, this kind of drugs can help inhibit generation of vessels of solid tumors, cause insufficient blood supply of tumor tissues and effectively inhibit tumors. In recent years, they are becoming increasingly recognized by scholars. More importantly, the anti-angiogenesis function will not be influenced by multi-drug resistance and can effectively exhibit a long-term antineoplastic activity.
The other advantage is that the chemotherapy drugs targeting colchicine site can effectively overcome drug resistance. Even though the antineoplastic drug targeting microtubule have exhibited strong activity in both separate administration and combined administration, its multi-drug resistance has limited its effect in the chemotherapy for the treatment of tumors. Currently, the resistance mechanism of taxol relates to three aspects: over-expression of MDR-1 gene, point mutations in α, β microtubule genes and expression of β-III tubulin monomer. Recently, US FDA approved epothilone's entry into the market, which is proved necessary to overcome the drug-resistance mechanism of β-III tubulin monomer. The clinical medication has demonstrated that the major drug-resistance mechanisms causing treatment failure of taxol and vinblastine drugs due to drug-resistance are over-expression of P-glycoprotein and altered expression of β-III tubulin monomer.
Structurally regarded as lactone compound generated through dehydration of cis-o-hydroxy cinnamic acid, coumarin compound is a general term for a kind of natural product containing parent nucleus of benzopyrone. Since Vauquelin firstly discovered the coumarin compound daphnin from plant Daphnealpina in 1812, hundreds of coumarin compounds have been obtained. The compounds widely exist in the plant kingdom, and particularly extensively in the Umbelliferae, Rutaceae, Asteraceae, Leguminosae, Solanaceae, etc. This component is contained in Chinese herbs such as Fructus Cnidii, Radix Angelicae Pubescentis, Radix Angelicae Dahuricae, Fructus Aurantii, Radix Peucedani, Ash Bark, Herba Artemisiae Scopariae, Fructus Psoraleae and Euphorbia Lathyris. Coumarin compounds have many obvious biological activities such as anti-viral, anti-tumor, anti-microbial, anti-cancer and anti-inflammatory activities, and have been attached with great importance by domestic and foreign scholars. Based on the difference of the substituent group on the parent nucleus and their locations, it can be divided into four categories: simple coumarins, furocoumarins, pyranocoumarins and other coumarins. Coumarin has fragrance and its representative compounds include angelica lactone, angelicone, xanthoxyletin, armillarisin A, etc.
Among the plural reported chemotherapy drugs, coumarin is verified to have minor or no toxicity through tests. Therefore, its action mechanism has aroused interests of many pharmacists. Currently, in the antitumor applications, coumarin is reported to have enzyme inhibitory activity, cell cycle arrest, anti-angiogenesi activity, heat shock protein (HSP90) inhibitory activity, telomerase inhibitory activity, anti-mitotic activity, carbonic anhydrase inhibitory activity, transport protein inhibitory activity, aromatase inhibitory activity and sulfatase inhibitory activity. Furthermore, scholars have conducted in-depth research into the structure-activity relationship of coumarin derivatives.
Tsyganov et al. have studied the anti-mitotic activity of coumarin compounds. They semi-synthesized 3-(4-methoxyphenyl) coumarin substituted by multi-alkoxy, and demonstrated that these compounds have anti-mitotic activity through phenotypic sea urchin embryo test. They also reported a compound A and pointed out that the source of the anti-mitotic activity of the compound A is related with the methoxyl at C5, C6, C7 positions of the parent nucleus of coumarin. The structure of trimethoxy truly appears in many microtubule inhibitors, such as colchicine and CA4. Meanwhile, they concluded from the study that coumarin with substituted aromatic group at 3 position is the characteristic of anti-mitotic drug of coumarins.
Biochemical Pharmacology, 2009, 77, 1773-1779 reported a coumarin compound B. The document revealed that the compound B can perform microtubule depolymerization and obviously cause cell arrest in G2/M phase, which conforms to the chemotherapy drugs targeting the colchicine site. Meanwhile, the IC50 for inhibiting tumor cells is between 44.8-475.2 nM, and that for inhibiting normal cells is greater than 5 μM. The compound B also demonstrates obvious inhibition for drug-resistant tumor cell strain. The compound B structurally has a diethylamino at C7 position to replace the methoxyl. Furthermore, as the same as compound A, the compound B also has an aromatic substitution structure at C3 position. The substitutive derivative at C3 position is deemed as the characteristic of microtubule inhibition drugs.
J. Med. Chem. 2011, 54, 3153-3162 reported a coumarin derivative compound C. The document revealed that the compound C has strong anti-tumor activity with the IC50 value of dozens of nanomolar. Meanwhile, it is discovered that the compound C can perform microtubule depolymerization, similar to colchicine and CA4. More importantly, the compound C still exhibits obvious inhibition to the drug-resistant strain with overexpressed P-glycoprotein. The report for the compound C has aroused everyone's attention to the coumarin with substituted aromatic group at C4 position and has expanded the modifiable scope of coumarin derivatives.
J. Med. Chem. 2009, 52, 2341˜2351 reported a compound D (MPC-6827, Azixa), whose IC50 value for plural tumor cells is between 1-10 nM. The compound D has entered into clinical phase II for the treatment of multiple neuroglioma and clinical phase I for the treatment of melanoma. Cancer Res. 2007 Jun. 15, 67(12): 5865˜71 reported that the functioning mechanism of the compound D is to target the colchicine site and inhibit polymerization of tubulin, so as to stop mitosis and induce cell apoptosis. In the document, the computer simulation results indicated that the 2-substituted functional group of the compound D occupied an important pocket of the colchicine site, and the bigger the 2-substituted group is, the lower the anti-microtubule activity is, wherein, the methyl or halogen atom (such as chlorine atom) substitution makes great contribution for maintaining the anti-microtubule activity of the compound D. No substitution at 2 position of the compound D will lead to total loss of anti-microtubule activity. Furthermore, the 1-position aromatic nitrogen atom and 3-position aromatic nitrogen atom of quinazoline have different functions in maintaining the anti-microtubule activity, wherein, the 1-position aromatic nitrogen atom forms hydrogen bond with the hydrogen bond donor in tubulin, which contributes to maintaining the activity of tubulin, while the 3-position aromatic nitrogen atom has no such function. Meanwhile, the substituent methyl of the nitrogen atom at 4 position of quinazoline is also important in inhibiting activity of tubulin. If the methyl is substituted by other groups, such as hydrogen, the anti-microtubule activity will lose as well. Although the compound D has demonstrated good anti-tumor activity, the phase I and II clinical trials have revealed great toxicity, which may limit its efficacy.
The structural formulas of the above compounds A, B, C and D are as follows:

Currently, it is of urgent need to develop safe and low toxic compounds which may effectively resist drug-resistance.